Simian Virus 40 T antigen has been used extensively to elucidate the functions necessary for virally induced tumorigenesis. The steps required to initiate and maintain tumorigenesis, however, are still vague. In cell culture, T antigen is both necessary and sufficient for the transformation of activity growing cells. Nonetheless, which of T antigen's multiple activities are sufficient to transform cells are as yet unknown. Functions such as binding to the tumor suppressor products, p53 and Rb (retinoblastoma gene product) have been strongly implicated in the transformation process. However, it appears in some instances, that these functions may be required but are not sufficient. The objective of this research proposal is to determine what contribution T antigen's transactivation activity plays in the transformation process. The specific goals of the study are to first investigate the existence of multiple transactivation activities in T antigen and assess if there is study are to first investigate the existence of multiple transactivation activities in T antigen and assess if there is a promoter specificity component. The transactivation will be assessed by using the secreted human placental alkaline phosphatase reporter gene (SEAP). T antigen fusion constructs, deletion and point mutants will be co-transfected with the reporter plasmid into the African Green monkey kidney cell line, TC7. The transactivation then will be evaluated by using a chemiluminescence assay. To determine if distinct transactivation regions of T antigen specific for either the RNA polymerase I verse RNA polymerase II promoters, the rat ribosomal promoter will be used to drive the SEAP gene. This construct will yield data to confirm the ability of T antigen to transactivate athe ribosomal promoter as well as localize the polypeptides necessary for the activity. For the RNA polymerase II construct the adenovirus E2 promoter will be used. The second objective of this research proposal is then to determine the relationship between transactivation and cellular transformation. Mutants will be investigated for their ability to transform rat embryo fibroblast in cooperation with an activated ras. Thus this study will attempt to dissociate athe transactivation and transformation (as assayed by ras cooperation) functions. This proposal is well within the ability and scope of the principle investigator and the facilities of Elizabethtown College. The chemiluminescence assay and ras cooperative transformation assay will provide a safe and sensitive methods for the training of undergraduate students.